Knock-out or knock-in? Converting a SacB-based gene disruption system for site-specific chromosomal integration in Pseudomonas syringae pv. tomato DC3000
Journal
Journal of Microbiological Methods
Journal Volume
145
Pages
50-58
Date Issued
2018
Author(s)
Abstract
Recent advances in next generation sequencing technology allow us to retrieve the whole genome sequence of a requested bacterium in less than a day. Thus, development of quick, easy and efficient means to systemically analyze the functions of all genes is required in the post-genome era. Here, a procedure of finding a suitable chromosome integration site and developing a gene disruption system into a knock-in system in Gram-negative bacteria is proposed. As a proof of concept, we successfully modified a sacB-based gene knock-out strategy into a site-specific gene integration system to deliver a DNA fragment into the genome site between 313,520 bp and 313,521 bp of the model phytopathogenic bacterium, Pseudomonas syringae pv. tomato (Pst) DC3000. The expression levels of avrPtoB and hcp2 integrated using this method exhibited steady and similar expression levels as those in the wild type. In the future, this concept could allow us to easily develop gene replacement and delivery systems at the same time using a counter-selectable suicide vector-based allelic exchange strategy, and facilitate functional genomics studies of any bacterium whose genome has been sequenced. ? 2018 Elsevier B.V.
Subjects
Genome engineering; pK18msLP; Pseudomonas syringae pv. tomato DC3000; Site-specific chromosomal integration
SDGs
Other Subjects
bacterial protein; avrPto protein, Pseudomonas syringae; bacterial DNA; bacterial protein; allele; Article; bacterial chromosome; bacterial gene; bacterial genome; bacterial strain; bacterial virulence; chromosomal localization; controlled study; DNA fragmentation; DNA modification; down regulation; functional genomics; gene delivery system; gene disruption; gene editing; gene expression; gene inactivation; gene insertion; gene location; gene targeting; gene vector; nonhuman; priority journal; protein expression; Pseudomonas syringae pv. tomato; sacB gene; wild type; bacterial chromosome; gene deletion; gene expression regulation; genetics; metabolism; microbiology; molecular cloning; pathogenicity; plant disease; plasmid; Pseudomonas syringae; tomato; virulence; Bacterial Proteins; Chromosomes, Bacterial; Cloning, Molecular; DNA, Bacterial; Gene Deletion; Gene Expression Regulation, Bacterial; Lycopersicon esculentum; Plant Diseases; Plasmids; Pseudomonas syringae; Virulence
Type
journal article